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ACOT2 / Acyl-coenzyme A thioesterase 2, mitochondrial · IHC design guide
Design Immunohistochemistry for ACOT2
Plan paraffin-section ACOT2 IHC around the cytoplasmic, mitochondrial-pattern staining reported in most tissues (HPA tissue IHC). The guide covers fixation, staining controls and interpretation, including the caution that staining may reflect proteins from more than one gene (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ACOT2 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic staining with a mitochondrial pattern (HPA tissue IHC) | |
| Staining pattern | Most tissues: cytoplasmic staining with a mitochondrial pattern (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet A10261-1); verify before use. | |
| Caveat | Staining may reflect proteins from more than one gene (HPA tissue IHC) | |
| Regulation | No specific expression regulator reported (UniProt) | |
| Isoform / epitope | Two isoforms; epitope differences are unreported (UniProt) |
Section 1
Recommended ACOT2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by one published ACOT2 brain-section IHC protocol (PMC11005155).
| Sample | Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A10261-1) |
| Fixation | Image fixative and duration unreported (datasheet A10261-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-ACOT2, 1:100-1:300 (datasheet A10261-1) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ACOT2-positive staining in endocrine cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression wit a mitochondrial pattern in most tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page antigen retrieval rule); the published protocol does not specify retrieval (PMC11005155).
Section 2
What Is the Expected ACOT2 Staining Pattern?
ACOT2 is mitochondrial and has no annotated transmembrane segment (UniProt P49753: location and topology). In paraffin IHC, expect cytoplasmic staining with a mitochondrial pattern, particularly in HPA high staining cells such as kidney collecting ducts and bronchial respiratory epithelium (HPA: tissue IHC). HPA rates the tissue profile Enhanced, but cautions that its antibodies target proteins from more than one gene (HPA: tissue IHC reliability).
| Fine granular cytoplasmic staining in collecting ducts or respiratory epithelium. | This fits the reported mitochondrial pattern and High staining in those cell types (HPA: tissue IHC). Individual mitochondria need not be resolved by chromogenic IHC (general IHC practice). |
| Predominantly nuclear, membranous or extracellular staining. | This conflicts with the mitochondrial location and lack of a transmembrane segment (UniProt P49753: location and topology). Check background and antibody specificity before calling it ACOT2 (general IHC practice). |
| Strong staining in adipocytes, with little staining in nearby expected cells. | HPA reports adipocytes as Not detected in adipose tissue (HPA: tissue IHC). Consider cross-reactivity or endogenous detection activity; the HPA multi-gene targeting caution prevents an ACOT2-specific assignment (HPA: reliability; general IHC practice). |
| Diffuse colour across cells and surrounding tissue. | A diffuse field obscures the reported cytoplasmic mitochondrial pattern (HPA: tissue IHC). Review no-primary controls and chromogen development for background before scoring cells (general IHC practice). |
| No staining in a kidney section that contains collecting ducts. | Collecting ducts are reported High by HPA (HPA: tissue IHC). First check section quality, retrieval and detection controls; a negative result alone cannot establish absent ACOT2 (general IHC practice). |
💡Expected ACOT2 appearanceCall a positive when expected cells show clear, fine granular cytoplasmic signal, often strong in HPA High cell types; dominant nuclear or extracellular colour is suspect (HPA: tissue IHC; UniProt P49753: location).
| Cell type within a tissue | Score named cells rather than the whole section: collecting ducts are High, whereas adipocytes in adipose tissue are Not detected (HPA: tissue IHC). |
| Tissue evidence across sources | UniProt lists strongest expression in heart, liver, muscle and kidney; HPA lists specific High-staining cells across several tissues. These describe different readouts, so use cell-level HPA IHC patterns for slide interpretation (UniProt P49753: tissue specificity; HPA: tissue IHC). |
| Antibody validation and specificity | HPA060170 has Enhanced IHC validation; HPA043705 is Supported (HPA: antibodies). HPA cautions that antibodies target proteins from more than one gene, so matching a pattern does not alone prove ACOT2 specificity (HPA: tissue IHC reliability). |
| Isoforms and processing | UniProt lists two isoforms and a 483-aa chain, with no annotated signal peptide or propeptide (UniProt P49753: isoforms and processing). The supplied sources give no antibody epitope map, so they cannot predict isoform-specific staining or an epitope retrieval requirement. |
| IF/ICC: what localisation is expected? | A mitochondrial pattern is supported by the approved HPA ICC-IF location, with images listed for U-251MG and U2OS (HPA: subcellular). HPA repeats its multi-gene antibody caution; use the separate IF/ICC guide for that application. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected cytoplasmic staining is absent in kidney collecting ducts. | The control cell population is HPA High, but absent colour can reflect an IHC workflow failure (HPA: tissue IHC; general IHC practice). | Confirm collecting ducts are present, then check section integrity, the assay's validated retrieval conditions and detection controls (general IHC practice). |
| Colour is mainly nuclear or extracellular. | That distribution disagrees with the reported mitochondrial location (UniProt P49753: location; HPA: tissue IHC). | Compare a no-primary control and an HPA High cell population; withhold an ACOT2-positive call if the discordant distribution persists (general IHC practice). |
| Adipocytes stain strongly. | Adipocytes in adipose tissue are reported Not detected; cross-reactivity or endogenous detection activity is possible (HPA: tissue IHC; general IHC practice). | Inspect no-primary and detection controls, then compare staining in named HPA High cells before interpreting specificity (general IHC practice). |
| Diffuse background hides cellular detail. | Nonspecific detection or overdeveloped chromogen can obscure a cellular pattern (general IHC practice). | Review blocking, washes and chromogen development against the assay controls; score only cells with distinguishable signal (general IHC practice). |
| Two antibodies produce different cell patterns. | HPA assigns different IHC validation statuses and cautions about multi-gene targeting (HPA: antibodies and tissue IHC reliability). | Record which antibody produced each pattern and compare named HPA cell types and controls; do not treat agreement with one image as proof of specificity (HPA: tissue IHC; general IHC practice). |
| Weak staining appears in a tissue expected to be strongly expressed. | UniProt's tissue-level expression summary does not specify which cells must stain strongly in an IHC section (UniProt P49753: tissue specificity). | Assess a named HPA High cell type in the same validated IHC workflow before judging the run; report the observed cell-level pattern (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing ACOT2 staining
A10261-1 · IHCImmunohistochemistry validation of ACOT2 using Anti-ACOT2 Antibody (A10261-1). Immunohistochemical analysis of paraffin-embedded human lung
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000119673-ACOT2/tissue/cerebral+cortex#imid_19917289
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000119673-ACOT2/tissue/colon#imid_19909207
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000119673-ACOT2/tissue/liver#imid_19909200
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000119673-ACOT2/tissue/kidney#imid_19909193
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody HPA043705; green = ACOT2, blue = nucleus, red = microtubules.
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody HPA043705; green = ACOT2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ACOT2 IHC & IF
🧪Run kidney first and require staining in collecting duct cells (HPA: High in kidney collecting ducts). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the kidney slide, count neighboring cells as internal negatives only if they lack specific chromogen above background (standard IHC practice).
Positive control tissue: Appendix (Endocrine cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ACOT2 in U-251MG, U2OS, with annotated localisation: Mitochondria (approved) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host-species- and clonality-matched isotype controls, alongside ACOT2-knockout tissue as a biological specificity control (standard IHC practice). Block endogenous peroxidase for chromogenic detection and check kidney endogenous biotin if using biotin-based detection (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the fixative is unreported in the selected A10261-1 paraffin-section caption (selected tissue-IHC caption). Antigen-retrieval dependence is unreported, so optimize retrieval empirically for paraffin sections (standard IHC practice). Whether frozen sections or IF are easier is unestablished; kidney endogenous biotin can complicate biotin-based detection (standard IHC practice).
HPA tissue IHC evidence for ACOT2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Endocrine cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | Glial cells | High | Protein (IHC) | HPA → |
| Duodenum | Endocrine cells | High | Protein (IHC) | HPA → |
| Kidney | Collecting ducts | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ACOT2 IHC Tips
Troubleshoot ACOT2 chromogenic IHC in paraffin sections using the stated retrieval method, tissue evidence, and mitochondrial localisation.
What retrieval conditions should I start with for ACOT2 IHC in paraffin sections?
Start with citrate buffer at pH 6.0, using heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval specification). Allow slides to cool in retrieval buffer before washing, and process comparison sections in the same run so differences in staining can be judged against identical heating (standard IHC practice). If staining is weak, compare a modestly longer heating time in the same buffer while watching for tissue damage or increased diffuse staining (standard IHC practice). Record retrieval time and temperature with each result, because the selected A10261-1 paraffin-section caption does not report retrieval conditions (A10261-1 caption).
How should I troubleshoot weak staining when fixation may be involved?
Target-specific fixation sensitivity for ACOT2 is unknown from the supplied evidence; the A10261-1 lung cancer caption identifies paraffin-embedded tissue but does not state its fixative (A10261-1 caption). Review the specimen's recorded fixative and fixation duration, then compare sections with different processing histories only after applying the same retrieval and detection conditions (standard IHC practice). If a well-preserved reference section stains while a similarly processed study section does not, assess tissue preservation, section adherence, and reagent performance before assigning a biological difference (standard IHC practice). Do not use the HPA staining pattern to infer how fixation affects this antibody (HPA tissue IHC; A10261-1 caption).
Where should convincing ACOT2 staining appear in a positive cell?
Expect cytoplasmic staining with a mitochondrial pattern, consistent with ACOT2's mitochondrial annotation and the HPA tissue profile (UniProt P49753; HPA tissue IHC). Judge the pattern within intact cells, using cellular morphology and the counterstain to distinguish cytoplasm from nuclei and extracellular material (standard IHC practice). Diffuse nuclear-only colour or deposit outside cells should prompt review of counterstain, section damage, and chromogen background before it is scored as ACOT2 (UniProt P49753 localisation; standard IHC practice). The HPA mitochondrial assignment carries a cross-gene antibody caution, so compartmental agreement supports interpretation but does not alone establish antibody specificity (HPA subcellular).
Could isoforms or epitope accessibility explain discordant ACOT2 IHC results?
ACOT2 has 2 annotated isoforms, but the supplied evidence does not identify the A10261-1 epitope or establish which isoforms it detects (UniProt P49753; A10261-1 caption). Check any available immunogen information before comparing this antibody with another reagent, and avoid assigning a stained cell to one isoform on chromogenic intensity alone (standard antibody validation practice). ACOT2 has no annotated transmembrane segment, while lysines 104 and 470 have reported modifications; neither fact demonstrates an effect on this antibody's tissue epitope (UniProt P49753). When patterns disagree, compare matched sections and localisation, then seek an independent specificity control rather than assuming an isoform explanation (standard IHC practice).
How can IF help assess a questionable chromogenic ACOT2 pattern?
On a separate IF/ICC preparation, multiplex ACOT2 with a mitochondrial marker and a marker for the expected cell type, then assess signal within individual cells (UniProt P49753 localisation; standard IF practice). Choose spectrally separated fluorophores and place the weaker signal in a channel with low specimen autofluorescence, checking single-label and unstained controls before interpreting overlap (standard IF practice). Because ACOT2 is mitochondrial and has no annotated transmembrane segment, permeabilise sufficiently to access an intracellular epitope, then optimise detergent exposure against cell morphology (UniProt P49753; standard IF practice). HPA reports mitochondrial IF localisation but cautions that its antibodies target proteins from multiple genes, so colocalisation alone cannot establish ACOT2 specificity (HPA subcellular).
What should I check when ACOT2 DAB staining looks diffuse or widespread?
Compare the suspect section with a no-primary control and inspect whether colour follows cells, vessels, tissue edges, or damaged areas (standard chromogenic IHC practice). Verify the peroxidase-block step and assess nonspecific secondary-reagent binding, since both can produce chromogenic signal unrelated to ACOT2 (standard chromogenic IHC practice). The A10261-1 caption reports a 1:100 antibody dilution in paraffin-embedded lung cancer; use it as a documented reference point while titrating signal against background in your tissue (A10261-1 caption; standard IHC practice). Broad cytoplasmic staining needs careful scrutiny because HPA describes expression in most tissues and warns of cross-gene antibody recognition (HPA tissue IHC).
How should I quantify ACOT2 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, then use an H-score combining intensity and percentage of positive cells or report the percentage positive by cell type (standard IHC quantification practice). For cell-rich or heterogeneous regions, positive-cell density per mm² can supplement intensity scoring if the sampled area is recorded (standard IHC quantification practice). Normalise comparisons to the same viable tissue area or the same number of eligible cells, and keep retrieval, detection, exposure to DAB, and counterstain consistent (standard IHC practice). Report cytoplasmic pattern separately from intensity because HPA describes a mitochondrial pattern and flags cross-gene antibody recognition (HPA tissue IHC).
How can I distinguish true ACOT2 staining from artefact?
Give greatest weight to staining in intact cell cytoplasm with a mitochondrial pattern, matching the annotated location and reported tissue pattern (UniProt P49753; HPA tissue IHC). Check cell identity against the section's morphology: HPA reports high staining in kidney collecting ducts and lung alveolar type I cells, whereas adipocytes in adipose tissue were not detected (HPA tissue IHC). Treat nuclear-only staining, edge enhancement, necrotic deposits, and signal in a no-primary control as reasons to investigate localisation, processing, or endogenous enzyme activity before calling a positive result (UniProt P49753 localisation; standard IHC practice). HPA's cross-gene antibody caution means a plausible pattern still requires an independent specificity control for a firm ACOT2 assignment (HPA tissue IHC).
Boster reagents
Best ACOT2 / Acyl-coenzyme A thioesterase 2, mitochondrial IHC Antibodies
A10261-1 has an IHC image of paraffin-embedded human lung cancer (catalog image caption). IF is listed, and human, mouse, and rat reactivity is reported; no IF image is supplied (catalog: applications, reactivity, image alts).
Real IHC data
Anti-ACOT2 Antibody
Cat # A10261-1
A10261-1 will render with its IHC image from paraffin-embedded human lung cancer (catalog image caption). Its listed applications include IHC and IF, with reported human, mouse, and rat reactivity; the payload supplies no IF image (catalog: applications, reactivity, image alts).
Which to pick: For tissue IHC, choose A10261-1: its own caption documents paraffin-embedded human lung cancer at 1:100, but does not report the fixative (catalog image caption). For IF, A10261-1 is listed at 1:200–1:1000; ICC and IF image validation are unreported (catalog: applications, IF dilution, image alts). It is also the listed option for human, mouse, and rat, with rabbit host and unspecified clonality; the supplied IHC image documents human tissue only (catalog: reactivity, host, clone, image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P49753 (ACOT2_HUMAN, Acyl-coenzyme A thioesterase 2, mitochondrial).
- Human Protein Atlas. ACOT2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ACOT2 subcellular location (ICC-IF): Localized to the mitochondria. Caution: Based on antibodies targeting proteins from multiple genes..
- Human Protein Atlas. ACOT2 antibody validation summary (2 antibodies).
- The discovery of regional neurotoxicity-associated metabolic alterations induced by carbon quantum dots in brain of mice using a spatial metabolomics analysis. Particle and fibre toxicology 2024 — PMC11005155.
- PubMed PMID:7596406 — UniProt-cited evidence.
- PubMed PMID:16940157 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ACOT2 (Acyl-coenzyme A thioesterase 2, mitochondrial). Expected localisation Cytoplasmic staining with a mitochondrial pattern (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A10261-1 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Endocrine cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A10261-1); 2. Fixation: Image fixative and duration unreported (datasheet A10261-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-ACOT2, 1:100-1:300 (datasheet A10261-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet A10261-1); verify before use.; Caveat: Staining may reflect proteins from more than one gene (HPA tissue IHC); Isoform / epitope: Two isoforms; epitope differences are unreported (UniProt); Regulation: No specific expression regulator reported (UniProt). Evidence: UniProt P49753, HPA tissue IHC · Enhanced, HPA ICC-IF · mitochondria. Caution: Based on antibodies targeting proteins from…, Datasheet A10261-1, PMC11005155. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ACOT2/immunohistochemistry-ihc-guide.